Polyamines are small, polycationic molecules with amino groups that are present in most living organisms. Studies indicate that polyamines increase general protein synthesis and are essential for efficient translation. While progress has been made in understanding the role of polyamines in translation in bacteria and mammals, their contribution and mode of action in plants remain largely unexplored. In a previous study, we found that putrescine (Put) and the pathogen-associated molecular pattern (PAMP) from bacterial flagellin (flg22) transcriptionally induced ribosome biogenesis in plants. Here we examined the impact of polyamines (Put and spermine, Spm) and flg22 on ribosome complex formation in Arabidopsis. Our results indicate that polyamines, flg22 and their combinations increase the abundance of actively translating polysomes. Riboproteomic analyses revealed that polyamines and flg22 trigger differential changes in the accumulation of ribosomal proteins, which are structurally confined in response to Put. Importantly, Put was found binding to non-translating and actively translating ribosomes, suggesting that this polyamine has a role in functional aspects of translation, such as stabilization and/or remodeling of polysomal complexes. Additional global proteomics analyses in polyamine biosynthesis mutants revealed that lower Put availability triggers changes in proteins associated with ribonucleoprotein complex binding and biogenesis. Overall, our findings highlight the effect of polyamines and flg22 on shaping the ribosomal protein composition of actively translating ribosomes in plants.

In temperate climates, low temperatures represent a significant stressor that adversely affects crop yield and production. Tomato (Solanum lycopersicum L.) is a subtropical crop cultivated in temperate regions. However, most tomato cultivars are sensitive to chilling temperatures, which limit their cultivation in colder regions. Some microorganism-based plant biostimulants have been reported to enhance abiotic stress tolerance in crops. In this study, we isolated two Pararhizobium sp. strains (44 and 128) and tested their potential to trigger chilling stress tolerance in tomato. Through transcriptional, metabolic and biochemical analyses we demonstrate that inoculation with strains 44 and 128 enhance chilling stress tolerance by stimulating the ICE1-CBF-COR cold stress signaling pathway at transcriptional level, improving reactive oxygen species (ROS) detoxifying capacity and boosting the biosynthesis of stress-protective metabolites, such as polyamines and reduced glutathione (GSH). Treatment of tomato plants with these strains under non-stress conditions also increased tomato fruit weight and quality attributes. These findings suggest that Pararhizobium strains 44 and 128 could be valuable biostimulants for improving chilling stress tolerance and crop yield.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r -proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context -dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context -dependent functionality of r -protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to highresolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot -gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r -proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r -proteins and normalize their intensities by the sum of r -protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r -protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r -proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r -proteins determined by stable isotope -assisted mass spectrometry. Our method purifies the r -proteins in a reversibly denatured state, which offers the possibility for combined top -down and bottom -up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r -proteins. As such, our method is better suited to conserve post -translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r -proteins to select an appropriate protease for digestion. We avoid nonspecific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r -proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non -labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r -protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top -down and bottom -up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto highresolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements.

Polyamines are small aliphatic polycations present in all living organisms. In plants, the most abundant polyamines are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamine levels change in response to different pathogens, including Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the regulation of polyamine metabolism and their specific contributions to defence are not fully understood. Here we report that stimulation of Put biosynthesis by Pst DC3000 is dependent on coronatine (COR) perception and jasmonic acid (JA) signalling, independently of salicylic acid (SA). Conversely, lack of Spm in spermine synthase (spms) mutant stimulated galactolipids and JA biosynthesis, and JA signalling under basal conditions and during Pst DC3000 infection, whereas compromised SA-pathway activation and defence outputs through SA-JA antagonism. The dampening of SA responses correlated with COR and Pst DC3000-inducible deregulation of ANAC019 expression and its key SA-metabolism gene targets. Spm deficiency also led to enhanced disease resistance to the necrotrophic fungal pathogen Botrytis cinerea and stimulated endoplasmic reticulum (ER) stress signalling in response to Pst DC3000. Overall, our findings provide evidence for the integration of polyamine metabolism in JA- and SA-mediated defence responses, as well as the participation of Spm in buffering ER stress during defence.

Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression ofENHANCED DISEASE SUSCEPTIBILITY(EDS1) andNONEXPRESSOR of PR GENES1(NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen-triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses.